Microparticles with enhanced covalent binding capacity and their uses

ABSTRACT

Disclosed are proteins which are covalently bound to a solid support at a first temperature where they have a first configuration, and then biomolecules are attached to the bound proteins at a higher temperature at which the proteins have more exposed functional groups, each such group being capable of covalently bonding to a biomolecule. The biomolecule can be, for example, a nucleic acid, including an amine functionalized oligonucleotide. The proteins can include, BSA (Bovine Serum Albumin) which can be bound using a reaction with the surface of a tosyl-activated microparticle.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/947,095, filed Sep. 22, 2004, now U.S. Pat. No. 7,595,279, whichclaims priority to U.S. Provisional Application No. 60/504,716, filed onSep. 22, 2003.

FIELD OF THE INVENTION

This invention is in the field of polyelectrolyte chemistry.

BACKGROUND

As an alternative to solve many of the problems associated withdiagnostic use of “spotted arrays” of oligonucleotides (the problems areoutlined in “Multianalyte Molecular Analysis Using Application-SpecificRandom Particle Arrays,” U.S. application Ser. No. 10/204,799, filed onAug. 23, 2002; WO 01/98765) preferred arrays are formed by bindingoligonucleotide probes to encoded microbead particles, including,encoded particles made of polymer resin. See U.S. patent applicationSer. No. 10/271,602 “Multiplexed Analysis of Polymorphic Loci byConcurrent Interrogation and Enzyme-Mediated Detection,” filed Oct. 15,2002, and Ser. No, 10/204,799 supra. The encoded particle-probeconjugates are then assembled in a 2D array format and placed in contactwith samples anticipated to contain target polynucleotides withsubsequences complementary to the probes, where the targetpolynucleotides in the samples were previously fluorescently labeled.Binding between the probes and targets is determined by the presence ofa fluorescent assay signal. Particular probes generating a positiveassay signal can be determined by decoding the array.

There are several known and commercially available methods forattachment of oligonucleotide probes to microbeads. A great number ofcovalent immobilization schemes for oligonucleotide probes tomicroparticles have been devised and are available either in openliterature or commercially. Traditional covalent immobilizationtechniques use functionalized beads (i.e, beads functionalized withreactive groups like amino, carboxyl, tosyl, aldehyde, epoxy, hydrazideand others) to link to complementary functional groups on the end ofoligonucleotide probes (Maire K. Walsh, Xinwen Wang and Bart C. Weimer,Optimizing the immobilization of single-stranded DNA onto glass beads,J. Biochem. Biophys. Methods 2001; 47:221-231). Often times such bindingprotocols lead to improper orientation and steric hindrance problems.The hybridization performance of such covalently immobilized probes canbe improved by introduction of spacer molecules (Edwin Southern, KalimMir and Mikhail Shchepinov; Molecular Interactions on Microarrays,Nature Genetics Supplement, 21, 1999, pp. 5-9), however, implementationis often difficult and impractical.

A practical and robust probe binding chemistry is therefore importantfor the optimal performance of a microbead array based assay. Thechemistry must allow the probes to bind to the particles with highefficiency, in order to maintain a consistent concentration of probes onthe bead surface and also the reaction must not alter the efficiency ofprobe-target binding. Moreover, the reaction must have minimum batch tobatch variability. In one commonly used method, functionalizedmicroparticles are coated with Neutravidin (Pierce, Rockford, Ill.),streptavidin or avidin, which are biotin binding proteins, to mediateimmobilization of biotinylated probes. The avidin-biotin interaction ishighly specific and one of the strongest known (with an associationconstant (K_(A)) of the order of 10¹⁵ M⁻¹ in aqueous solutions) andprovides nearly irreversible linkage between the bead surfaceimmobilized protein and the biotinylated probe molecule. See U.S. patentapplication Ser. No. 10/271,602, supra. The method described below forbinding probes to polyelectrolytes are preferred to these known methods,because they were demonstrated as capable of inducing attachment ofgreater numbers of oligonucleotides to beads.

SUMMARY

A polyelectrolyte having multiple exposed functional groups, each suchgroup being capable of covalently bonding to a molecule, is immobilizedon a surface for the purpose of bonding to a biomolecule. Thebiomolecule can be, for example, a nucleic acid, e.g., an aminefunctionalized oligonucleotide. The polyelectrolyte can include, e.g.,BSA (Bovine Serum Albumin) which is bound to a functionalized surfaceusing a covalent immobilization strategy, e.g., reaction with thesurface of a tosyl-activated microparticle. Following such reaction,exposed reactive functional groups on the protein, such as amine,carboxyl, thiol, hydroxyl groups can further be utilized to covalentlycouple the oligonucleotide of interest using suitable chemistry.

In one embodiment oligonucleotides modified at a terminal position (the3′ or 5′ terminal position) with amines (e.g., amino modifiedoligonucleotides) are covalently bound to BSA using an EDAC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) reaction (see, e.g., D.Seligal et al., Analytical Biochemistry 218:87091 (1994)). The covalentreaction results in the formation of an amide bond between the aminegroup at the terminus of the oligonucleotide and carboxyl groups on theBSA. The reaction is illustrated in FIG. 1.

The functionalized surface can be the surface of a bead ormicroparticle, which can be composed of any of a number of materials,including polymers, polymer resins, glass, latex or others which can befunctionalized for immobilization of a polyelectrolyte. Experiments wereperformed comparing BSA-coated beads with human serum albumin (“HSA”)another exemplary polyelectrolyte, and with Neutravidin as well. Theresults of hybridization experiments indicated that the BSA-coated beadswere capable of attaching greater concentrations of oligonucleotides tothe beads.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the bonding of BSA to functionalized beads and thebonding of an oligonucleotide probe to the BSA using an EDAC reaction.

FIG. 2 shows the hybridization signals from oligo-functionalized BSAcoupled beads as a function of the amount of added aminated probe forcoupling. A perfectly matching probe was attached to two sets ofBSA-coupled beads. BSA was coupled to the first set of beads at 65° C.and to a second set at 37° C. A much higher hybridization efficiency wasnoted (higher signal) on the first set of beads to which BSA was coupledat 65° C. A third set of beads coupled with BSA at 65° C. andfunctionalized with a mismatched negative control probe shows negligiblehybridization, thus indicating that the enhanced signal is not a resultof increased non-specific binding.

FIG. 3 shows titration results of BSA coupled beads. As in FIG. 2,efficiency of hybridization is greater for the beads coupled with BSA ata higher temperature than at a lower temperature, as demonstrated by thedifference in hybridization signal from a target placed in contact withan oligonucleotide probe bound to BSA-coupled beads where BSA wascoupled to one set of beads at 37° C. and where BSA was coupled toanother set of beads at 65° C. (see Example 4)

FIG. 4 indicates a differences in coupling efficiency of BSA to tosylfunctionalized beads at different temperatures, as determined using ahybridization assay, where oligonucleotide probes are bound to the BSAimmobilized on the beads and then reacted with a complementaryfluorescently labeled target, (see Example 6)

FIG. 5 indicates that for incubation at 65° C. or higher for about 1hour, for the coupling reaction of BSA to tosyl activated beads, thebinding efficiency of BSA to the bead surface is not affected, asdemonstrated by the difference in hybridization signal from a targetplaced in contact with an oligonucleotide probe bound to BSA-coupledbeads. (see Example 7)

FIG. 6A shows that BSA coated tosyl functionalized beads give a moreuniform and stronger hybridization signal, following bonding of probesand hybridization with a target than a Neutravidin-coated tosyl bead.(see Example 8)

FIG. 6B shows the coefficient of variation of the signals in FIG. 6A,

FIG. 7 shows a significant difference in hybridization when HSA, ratherthan BSA, is the polyelectrolyte coated on tosyl functionalized beads,where oligonucleotide probes are bound, respectively, to BSA or HSAimmobilized on beads, and then reacted with a complementaryfluorescently labeled target.

DETAILED DESCRIPTION Example 1 Preparation of BSA-Coated TosylFunctionalized Beads

BSA solution at the concentration of 5 mg/mL is prepared by dissolving50 mg of BSA in 10 mL of PBS. 2.0 mL of PBS-T is added to a 15 mLcentrifuge tube. 1 mL of fluorescence colored beads at the concentrationof 1% solids (10 mg) are transferred into the centrifuge tube, and mixedwell by vortexing. The beads are spun down by centrifugation at 3,500rpm for 4+/−0.5 minutes, and the supernatant is decanted. The beads arere-suspended by adding 3.0 mL of PBST into the tube, and mixed well byvortexing. The beads are again spun down by centrifugation at 3,500 rpmfor 4+/−0.5 minutes, and the supernatant is discarded. 3.0 mL of BSAsolution (5 mg/mL) are added to the beads, and mixed well by vortexing.The tubes are placed on a shaker in a 37° C. incubator, and the beadsare allowed to react overnight with mixing at 250 rpm.

Thereafter, the beads are spun down by centrifugation at 3,500 rpm for 4minutes, and the supernatant is discarded. The beads are then washed byadding 3.0 mL of PBS-T to the tube, and mixed on a vortex mixer. Thebeads are then again centrifuged at 3500 rpm for 4+/−0.5 minutes, andthe supernatant is poured off. The washing and centrifuging steps arethen repeated.

3.0 mL of storage buffer (0.1 M PBS containing 0.1% NaN₃), are added,and mixed on a vortex mixer. The beads are again centrifuged at 3,500rpm for 4+/−0.5 minutes, and the supernatant is poured off. The beadsare then resuspended in 1 ml of storage buffer by vortexing. The beadsare at a concentration of 1% solids (10 mg/mL), and are stored at 4-6°C. They are ready for attachment of amine-containing biomaterials (e.g.,BSA) through the EDAC reaction, as described below in Example 3.

Example 2 Preparation of BSA-Coated Carboxyl Functionalized Beads

The coupling of BSA to carboxylated particles is carried out as follows.100 μl of carboxylated particles at a concentration of 1% solids istransferred to a 2 ml Eppendorf tube. The beads are then pelleted bycentrifugation and the supernatant removed. Following this, the beadsare washed 1× with 1 ml of MES (details) buffer (pH 4.5). Separately astock solution of BSA (5 mg BSA/ml) in MES buffer and EDC (20 mg/ml) inMES buffer are prepared, 100 μl of the BSA stock solution is added tothe bead pellet and the suspension mixed well by vortexing. Followingthis, 400 μl of the EDC stock solution is added to the bead suspension,mixed well by vortexing and allowed to react a room temperature for 1 hrwith end-over-end mixing. After 1 hr incubation, 100 μl of PBS-T isadded to the suspension and the beads centrifuged. The pellet is washedtwice with 1 ml PBS-T by centrifugation-redispersion cycle, and thebeads are finally suspended in 100 μl of storage buffer (0.1 M PBScontaining 0.1% sodium azide, NaN₃) and stored at 4-6° C.

Example 3 EDAC Reaction for Coupling of Aminated Oligonucletide Probesto BSA Beads

The coupling of aminated oligonucletide probes to the beads, prepared asin Example 1 and 2, was carried out as follows A series of 1.5 mlEppendorf tubes were taken and labeled to identify the type ofmicroparticle and the oligonucletide probe to be coupled. Followingthis, 500 μL of PBST was dispensed into each tube, followed by 100 μL ofBSA coupled beads at concentration of 1% solids. The tubes were mixedwell with a vortex mixer for 10 seconds. The beads were then spun downat 9500 rpm for 2+/−0.5 nm, and the supernatant discarded. A 500 μLaliquot of 0.05 M MES buffer (pH4.5) was added to the pellet, and mixedwell by vortexing. The beads were then centrifuged at 9500 rpm for2+/−0.5 minutes, and the supernatant discarded. A 500 ul aliquot of 0.05M of EDAC in MES buffer (prepared right before use) was added to thebeads, and mixed well by vortexing. Following 10 μL each of aminomodified DNA probes (e.g., probe MS-508 N25, purchased from IntegratedDNA Technologies, Inc., Coralville Iowa) was added at a concentration of100 μM to each of the tubes containing the bead suspensions, and mixedwell. The reaction is allowed to proceed for 1 hour at room temperature(20-25° C.) with end-over-end mixing.

After the incubation, 100 μL PBS-T is added to each tube, and mixed byvortexing. The beads are then spun down in a centrifuge at 9500 rpm for2+/−0.5 minutes, and the supernatant discarded. The beads are thenwashed twice with 500 ul PBS-T using the centrifugation redispersioncycle.

The beads are resuspended in 100 μL of PBST to bring the finalconcentration to 1% solids, and stored at 4-6° C. for further use.

The hybridization performance (see Example 4 for protocol) ofoligonucleotide functionalized particles as a function of added amountof oligo (0.25, 0.5, 1, 2, 4, 8 ul of 100 uM/200 ug particles) is shownin FIG. 2. The amount described above 10 ul of 10 uM/1 mg thusrepresents a saturation concentration. Also, the beads with BSA coupledat higher temperature show improved hybridization performance asdescribed in detail later.

Example 4 Hybridization Assay Using Oligonucletide Functionalized Beads

-   1. Bead mixtures are assembled on 8 different chips. Stock    fluorescently labeled DNA target solution (MS508-90mer-CY5) is    prepared in hybridization buffer (1×TMAC. Eight different serial    dilutions are prepared from the stock target solution. 20 μl of each    of the serially diluted target solutions are then added to the eight    separate chips.-   2. A slide, containing the chips, is placed in a hybridization    heater/shaker, and incubated at 55° C. for 20 minutes at 100 rpm.-   3. The slide is removed and cooled to room temperature, and the    hybridization solution is removed with the transfer pipette.-   4. 20 μl of 1×TMAC is added to each chip, and the chip is washed by    pipetting the solution 8 to 10 times.-   5. The washing solution is removed and 5 ml of mounting solution    (1×TMAC) is added to each chip, and the assay signal (CY5) is read    under a fluorescent microscope using a coverslip.-   6. A titration curve is plotted of the hybridization signal (CY5) vs    DNA probe concentration.    Example of titration curves are shown in FIG. 3.

Example 6

Experiments were conducted to compare the effect of adding EDAC to thebead-probe suspension twice (EDAC is known to hydrolyze very quickly atacidic pH) to assess whether this leads to an enhanced binding of probesto the BSA layer. First, the probe MS-508-N25 was coupled to BSA-coatedbeads under each of the following condition; (10 μl 100 μM probe/100 μl1% beads). One-half of the beads were removed from the 1× tube after onehour of reaction time, and fresh EDAC was added, and then the reactionproceeded in this tube for one additional hour. The whole process wasthen repeated for the non-matching probe SSP 36. Each set of beads werepooled with the non-specific beads and assembled on a chip, and then allsets were placed in contact with target MS 508-40mer-Cy5 underhybridizing conditions. Results were then recorded, and are summarizedbelow in Table II. 2×EDAC addition provided higher hybridizationsignals.

TABLE II Model Non- Probe Assay specific Concentration Cy5 Signal CV Cy5Signal CV 1X EDAC 536.1 0.17 79.9 0.26 2X extra EDAC 732.9 0.17 53.30.19

Example 6 BSA Coupling to Tosyl Activated Beads at DifferentTemperatures and their Hybridization Characteristics

2.0 mL of PBST was added to each of five 15 mL centrifuge tubes and 1 mLof fluorescence colored beads, at the concentration of 1% solids (10mg), was added to each tube, and then the beads were mixed by vortexing.The beads were spun down by centrifugation at 3,500 rpm for 4+/−0.5minutes, and the supernatant was decanted. The beads were thenresuspended in 3.0 mL of PBST, mixed well by vortexing, and again spundown by centrifugation at 3,500 rpm for 4+/−0.5 minutes. The supernatantwas then poured off.

2 mL of PBS (pH7.2) and 1 mL of BSA solution (50 mg/mL in PBS) was addedto each tube, and mixed well by vortexing. The ambient temperature in anincubator for each of the tubes was set as follows: tube A—22° C. tubeB—37° C., tube—50° C., tube D—65° C. and tube E—75° C., and the beadswere allowed to react with BSA for 14 hours at the designatedtemperature, with end-over-end mixing. The tubes were then cooled toroom temperature, and the beads spun down by centrifugation at 3,500 rpmfor 4 minutes, and the supernatant poured off. The beads were thenwashed by adding 3.0 mL of PBST to the tube mixed on a vortex mixer, andspun down at 3500 rpm for 4+/−0.5 minutes. The supernatant was pouredoff.

1 mL of storage buffer (PBS containing 0.1% NaN₃) was added, and thetubes were mixed on a vortex mixer. The bead concentration was 1% solids(10 mg/mL). The BSA coupled beads were stored at 4-6° C.

The 25-mer MS-508 N25 biotinylated oligonucleotide probe was conjugatedto each set of beads through the EDAC coupling method described above.Each set of beads was then contacted with a fixed concentration oflabeled target (a 90-mer oligonucleotide labeled with Cy-5) for theprobe under hybridizing conditions. The quantity of label on the beadscorrelates with the probe concentration on the beads.

As shown in FIG. 4, the beads which were coupled to BSA at highertemperatures displayed more target binding to the oligonucleotide probesdisplayed on the bead surface. This indicates that there is a greaterconcentration of probes at the surface of such beads, which may bebecause at 65° C., BSA denatures and opens up, presenting more availablebinding sites to the probes.

Example 7 Comparison of Varying Incubation Time for BSA Coupling toTosyl Functionalized Particles

An Experiment was conducted to study the time course of BSA couplingreaction on tosylated particles. Following the same protocol as inExamples 1 and 5 above, 12 separate tubes, each containing a BSA-tosylparticle reaction mixture, were incubated at 65° C. in an oven, and onecontrol tube was incubated at 37° C. Each tube was taken out after apredetermined incubation period, washed and then coupled with aoligonucleotide probe (including one control probe) following methodoutlined in Example 3. Following this, a hybridization reaction wasperformed and the assay intensity was recorded (see Example 4). Theresults are shown in FIG. 5 which illustrates that the BSA couplingreaction is essentially complete in less than one hour.

Example 8 Comparison with Conventional Biotin-Avidin OligonucleotideCoupling and NeutrAvidin Coating Chemistry

An experiment was carried out to compare the capture and hybridizationefficiency of oligo-conjugated, BSA-functionalized beads withbiotinylated oligo-conjugated NeutrAvidin bead. The proteins werecoupled to the bead surface at 37° C. using a protocol as outlined inExample 1. Following this, biotinylated (and also aminated) oligos wereconjugated to particles (as in Example 3) and a hybridization assay wascarried out with a cognate target.

Two differently encoded but otherwise identical BSA coated particleswere taken and a matching probe was bound to one group and anon-matching probe was bound to the other group. Similarly two otherNeutrAvidin-functionalized beads were taken and bound to matched andmismatched biotinylated probes.

The results of the assay are shown in FIGS. 6A and 6B. It is evidentthat BSA coating provides a more uniform (lower CV) and higher signal tonoise ratio (the hybridization intensity on the mismatched probe wasconsidered as noise) than achieved when using the NeutrAvidin capturechemistry.

Example 9 Comparison with HSA Coating

HSA (Human Serum Albumin) was coupled under identical conditions tothose used for BSA coupling to tosyl-functionalized particles. The HSAfunctionalized particles were then coupled with oligonucleotide probesand hybridized (titrated) to a fluorescently labeled model DNA target(as in Example 4). The results are shown in FIG. 7. It indicates thatthe HSA coating is not as effective as the BSA coating for binding theoligonucleotide probes notwithstanding the fact that, like BSA, HSA hasmany functional carboxyl groups available for binding to theoligonucleotide probes.

Example 10 Batch to Batch Variation of BSA Coupling

Three batches of beads of 10 mg/each were separately coupled with BSA at65° C. for 14 hours, where the BSA-bead ratio was 5 (W/W, mg/mg). Thereaction volume for coupling was 3 mL. One batch of beads was coupled toBSA at 37° C. for use as a control. The coupling efficiency wasdetermined based on signal intensity for hybridization of DNA probescoupled to the beads to cognate targets. The hybridization was done at55° C. for 20 minutes in 1×TMAC and the target was MS508-90mer-CY5 at aconcentration of 400 nM. The integration time for assay read-out is 200ms. The results are shown in Table I.

TABLE I Batch CY5 Intensity (100 ms) 1 6864 2 6515 3 6431 Control 3964The 65° C. batches had a consistently higher intensity than the batchcoupled at 37° C. and also the batch to batch variability was small.

The terms, expressions and examples hereinabove are exemplary only, andnot limiting, and the invention is defined only in the claims whichfollow and includes all equivalents of the subject matter of the claims.

1. A product comprising: (i) a microparticle; (ii) a single layer ofbovine serum albumin molecules covalently attached to the microparticle;(iii) oligonucleotides covalently attached to the single layer whereinattachment of the single layer to the microparticle is performed at apre-selected temperature such that the bovine serum albumin molecules insaid single layer attached at said pre-selected temperature display agreater number of available sites for covalent attachment ofoligonucleotides than if the single layer was attached to themicroparticle at a different temperature.
 2. The product of claim 1wherein the pre-selected temperature is about 65° C.
 3. The product ofclaim 1 wherein the oligonucleotides are bound to the protein moleculesthrough an amide linkage, formed by an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide reaction.
 4. The product of claim 1 wherein the surface ofthe microparticle is activated with tosyl functional group.
 5. Theproduct of claim 1 wherein the oligonucleotide is DNA, RNA, a peptidenucleic acid, a locked nucleic acid, or mixtures thereof.
 6. The productof claim 1 wherein the oligonucleotide is modified at its 3′ or 5′ endwith a functional group.
 7. The product of claim 6 wherein thefunctional group is a primary amino group.